PDF(Pubmed)
Abstract：
Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1 U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance.
摘要：
细胞表面和可溶性胞外糖胺聚糖均已显示干扰非病毒基因递送的外源核酸递送效率，包括脂质复合物和多聚复合物介导的转染。商业上和临床试验中使用的大多数基因治疗病毒载体目前是使用基于瞬时转染的生物过程制造的。对病毒载体产品日益增长的需求，加上全球生产能力短缺，需要改进的转染技术和工艺，以最大限度地提高工艺效率和生产率。发现可溶性细胞外糖胺聚糖在悬浮适应的HEK293T细胞培养物的条件细胞培养基中积累，损害转染性能和慢病毒载体生产。特定的酶降解，硫酸软骨素，发现具有软骨素酶ABC的糖胺聚糖显着增强转染性能。此外,我们报道,与对照慢病毒载体生物过程中使用的细胞密度相比,当以更高的细胞密度培养细胞时,功能性慢病毒载体滴度显著改善;当转染前培养物补充软骨素酶ABC时,这种改善进一步增强.与现有方法相比，当转染前将细胞密度加倍时，计算出功能性慢病毒载体滴度增加71.2％，并且用0.1U/mL软骨素酶ABC处理高密度细胞培养物导致滴度进一步增加18.6％。提出了一种能有效提高转染性能的方法。
